Alpha-amylase variants

ABSTRACT

The invention relates to a variant of a parent Termamyl-like alpha-amylase, which variant exhibits altered properties, in particular reduced capability of cleaving a substrate close to the branching point, and improved substrate specificity and/or improved specific activity relative to the parent alpha-amylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 09/537,168filed Mar. 29, 2000, now U.S. Ser. No 6,410,295 issued Jun. 25, 2002 andclaims, under 35 U.S.C. 119, priority of Danish application no. PA 199900437 filed Mar. 30, 1999, and claims the benefit of U.S. provisionalNo. 60/127,427 filed on Apr. 1, 1999 the contents of which are fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, inter alia, to novel variants of parentTermamyl-like alpha-amulases, notably variants exhibiting alteredproperties, in particular altered cleavage pattern (relative to theparent) which are advantageous with respect to applications of thevariants in, in particular, industrial starch processing (e.g., starchliquefaction or saccharification).

BACKGROUND OF THE INVENTION

Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1)constitute a group of enzymes which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides.

There is a very extensive body of patent and scientific literaturerelating to this industrially very important class of enzymes. A numberof alpha-amylase such as Termamyl-like alpha-amylases variants are knownfrom, e.g., WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873, WO96/23874 and WO 97/41213.

Among recent disclosure relating to alpha-amylases, WO 96/23874 providesthree-dimensional, X-ray crystal structural data for a Termamyl-likealpha-amylase, reffered to as BA2, which consists of the 300 N-terminalamino acid residues of the B. amytoliquefaciens alpha-amylase comprisingthe amino acid sequence shown in SEQ ID NO: 6 herein and amino acids301-483 of the C-terminal end of the B. licheniformis alpha-amylasecomprising the amino acid sequence shown in SEQ ID NO: 4 herein (thelatter being available commercially under the tradename Termamyl™), andwhich is thus closely related to the industrially important Bacillusalpha-amylases (which in the present context are embraced within themeaning of the term “Termamyl-like alpha-amylases”, and which include,inter alia, the B. licheniformis, B. amytoliquefaciens and B.stearothermophilus alpha-amylases). WO 96/23874 further describesmethodology for designing, on the basis of an analysis of the structureof a parent Termamyl-like alpha-amylase, variants of the parentTermamyl-like alpha-amylase which exhibit altered properties relative tothe parent.

WO 96/23874 and WO 97/41213 (Novo Nordisk) discloses Termamyl-likealpha-amylase variants with an altered cleavage pattern containingmutations in the amino acid residues V54, D53, Y56, Q333, G57 and A52 ofthe sequence shown in SEQ ID NO: 4 herein.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates to novel alpha-amylolytic variants(mutants) of a Termamyl-like alpha-amylase, in particular variantsexhibiting altered cleavage pattern (relative to the parent), which areadvantageous in connection with the industrial processing of starch(starch liquefaction, saccharification and the like).

The inventors have surprisingly found variants with altered properties,in particular altered cleavage pattern which have improved reducedcapability of cleaving an substrate close to the branching point, andfurther have improved substrate specificity and/or improved specificactivity, in comparison to the WO 96/23874 and WO 97/41213 (NovoNordisk) disclosed Termamyl-like alpha-amylase variants with an alteredcleavage pattern containing mutations in the amino acid residues V54,D53, Y56, Q333, G57 and A52 of the sequence shown in SEQ ID NO: 4herein.

The invention further relates to DNA constructs encoding variants of theinvention, to composition comprising variants of the invention, tomethods for preparing variants of the invention, and to the use ofvariants and compositions of the invention, alone or in combination withother alpha-amylolytic enzymes, in various industrial processes, e.g.,starch liquefaction, and in detergent compositions, such as laundry,dish washing and hard surface cleaning compositions; ethanol production,such as fuel, drinking and industrial ethanol production; desizing oftextiles, fabrics or garments etc.

Nomenclature

In the present description and claims, the conventional one-letter andthree-letter codes for amino acid residues are used. For ease ofreference, alpha-amylase variants of the invention are described by useof the following nomenclature:

Original amino acid(s):position(s):substituted amino acid(s)

According to this nomenclature, for instance the substitution of alaninefor asparagine in position 30 is shown as:

Ala30Asn or A30N

a deletion of alanine in the same position is shown as:

Ala30* or A30*

and insertion of an additional amino acid residue, such as lysine, isshown as:

-   -   *30aLys or *30aK

A deletion of a consecutive stretch of amino acid residues, such asamino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33) ordelta(A30-N33).

Where a specific alpha-amylase contains a “deletion” in comparison withother alpha-amylases and an insertion is made in such a position this isindicated as:

*36aAsp or *36aD

for insertion of an aspartic acid in position 36

Multiple mutations are separated by plus signs, i.e.:

Ala30Asp+Glu34Ser or A30N+E34S

representing mutations in positions 30 and 34 substituting alanine andglutamic acid for asparagine and serine, respectively. Multiplemutations may also be separated as follows, i.e., meaning the same asthe plus sign:

Ala30Asp/Glu34Ser or A30N/E34S

When one or more alternative amino acid residues may be inserted in agiven position it is indicated as

A30N, E or

A30N or A30E

Furthermore, when a position suitable for modification is identifiedherein without any specific modification being suggested, or A30X, it isto be understood that any amino acid residue may be substituted for theamino acid residue present in the position. Thus, for instance, when amodification of an alanine in position 30 is mentioned, but notspecified, or specified as “X”, it is to be understood that the alaninemay be deleted or substituted for any other amino acid, i.e., any oneof: R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.

DETAILED DISCLOSURE OF THE INVENTION

The Termamyl-Like Alpha-Amylase

It is well known that a number of alpha-amylases produced by Bacillusspp. are highly homologous on the amino acid level. For instance, the B.licheniformis alpha-amylase comprising the amino acid sequence shown inSEQ ID NO: 4 (commercially available as Termamyl™) has been found to beabout 89% homologous with the B. amyloliquefaciens alpha-amylasecomprising the amino acid sequence shown in SEQ ID NO: 6 and about 79%homologous with the B. stearothermophilus alpha-amylase comprising theamino acid sequence shown in SEQ ID NO: 8. Further homologousalpha-amylases include an alpha-amylase derived from a strain of theBacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all ofwhich are described in detail in WO 95/26397, and the #707 alpha-amylasedescribed by Tsukamoto et al., Biochemical and Biophysical ResearchCommunications, 151 (1988), pp. 25-31.

Still further homologous alpha-amylases include the alpha-amylaseproduced by the B. licheniformis strain described in EP 0252666 (ATCC27811), and the alpha-amylases identified in WO 91/00353 and WO94/18314. Other commercial Termamyl-like B. licheniformis alpha-amylasesare Optitherm™ and Takatherm™ (available from Solvay), Maxamyl™(available from Gistbrocades/Genencor), Spezym AA™ and Spezyme Delta AA™(available from Genencor), and Keistase™ (available from Daiwa).

Because of the substantial homology found between these alpha-amylases,they are considered to belong to the same class of alpha-amylases,namely the class of “Termamyl-like alpha-amylases”.

Accordingly, in the present context, the term “Termamyl-likealpha-amylase” is intended to indicate an alpha-amylase, which at theamino acid level exhibits a substantial homology to Termamyl™, i.e., theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 4 herein. In other words, a Termamyl-like alpha-amylase is analpha-amylase, which has the amino acid sequence shown in SEQ ID NO: 2,4, 6, or 8 herein, and the amino acid sequence shown in SEQ ID NO: 1 or2 of WO 95/26397 or in Tsukamoto et al., 1988, or i) which displays atleast 60%, preferred at least 70%, more preferred at least 75%, evenmore preferred at least 80%, especially at least 85%, especiallypreferred at least 90%, even especially more preferred at least 95%homology, more preferred at least 97%, more preferred at least 99% withat least one of said amino acid sequences and/or ii) displaysimmunological cross-reactivity with an antibody raised against at leastone of said alpha-amylases, and/or iii) is encoded by a DNA sequencewhich hybridises to the DNA sequences encoding the above-specifiedalpha-amylases which are apparent from SEQ ID NOS: 1, 3, 5 and 7 of thepresent application and SEQ ID NOS: 4 and 5 of WO 95/26397,respectively.

In connection with property i), the “homology” may be determined by useof any conventional algorithm, preferably by use of the GAP program fromthe GCG package version 7.3 (June 1993) using default values for GAPpenalties, which is a GAP creation penalty of 3.0 and GAP extensionpenalty of 0.1, (Genetic Computer Group (1991) Programme Manual for theGCG Package, version 7, 575 Science Drive, Madison, Wis. USA 53711).

A structural alignment between Termamyl and a Termamyl-likealpha-amylase may be used to identify equivalent corresponding positionsin other Termamyl-like alpha-amylases. One method of obtaining saidstructural alignment is to use the Pile Up programme from the GCGpackage using default values of gap penalties, i.e., a gap creationpenalty of 3.0 and gap extension penalty of 0.1. Other structuralalignment methods include the hydrophobic cluster analysis (Gaboriaud etal., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading(Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).Property ii) of the alpha-amylase, i.e., the immunological crossreactivity, may be assayed using an antibody raised against, or reactivewith, at least one epitope of the relevant Termamyl-like alpha-amylase.The antibody, which may either be monoclonal or polyclonal, may beproduced by methods known in the art, e.g., as described by Hudson etal., Practical Immunology, Third edition (1989), Blackwell ScientificPublications. The immunological cross-reactivity may be determined usingassays known in the art, examples of which are Western Blotting orradial immunodiffusion assay, e.g., as described by Hudson et al., 1989.In this respect, immunological cross-reactivity between thealpha-amylases having the amino acid sequences SEQ ID NOS: 2, 4, 6, or8, respectively, have been found.

The oligonucleotide probe used in the characterization of theTermamyl-like alpha-amylase in accordance with property iii) above maysuitably be prepared on the basis of the full or partial nudeotide oramino acid sequence of the alpha-amylase in question.

Suitable conditions for testing hybridization involve presoaking in5×SSC and prehybridizing for 1 hour at ˜40° C. in a solution of 20%formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50mg of denatured sonicated calf thymus DNA, followed by hybridization inthe same solution supplemented with 100 mM ATP for 18 hours at ˜40° C.,followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40°C. for 30 minutes (low stringency), preferred at 50° C. (mediumstringency), more preferably at 65° C. (high stringency), even morepreferably at ˜75° C. (very high stringency). More details about thehybridization method can be found in Sambrook et al., Molecular_Cloning:A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.

In the present context, “derived from” is intended not only to indicatean alpha-amylase produced or producible by a strain of the organism inquestion, but also an alpha-amylase encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate analpha-amylase, which is encoded by a DNA sequence of synthetic and/orcDNA origin and which has the identifying characteristics of thealpha-amylase in question. The term is also intended to indicate thatthe parent alpha-amylase may be a variant of a naturally occurringalpha-amylase, i.e. a variant, which is the result of a modification(insertion, substitution, deletion) of one or more amino acid residuesof the naturally occurring alpha-amylase.

Parent Hybrid Alpha-amylases

The parent alpha-amylase may be a hybrid alpha-amylase, i.e., analpha-amylase, which comprises a combination of partial amino acidsequences derived from at least two alpha-amylases.

The parent hybrid alpha-amylase may be one, which on the basis of aminoacid homology and/or immunological cross-reactivity and/or DNAhybridization (as defined above) can be determined to belong to theTermamyl-like alpha-amylase family. In this case, the hybridalpha-amylase is typically composed of at least one part of aTermamyl-like alpha-amylase and part(s) of one or more otheralpha-amylases selected from Termamyl-like alpha-amylases ornon-Termamyl-like alpha-amylases of microbial (bacterial or fungal)and/or mammalian origin.

Thus, the parent hybrid alpha-amylase may comprise a combination ofpartial amino acid sequences deriving from at least two Termamyl-likealpha-amylases, or from at least one Termamyl-like and at least onenon-Termamyl-like bacterial alpha-amylase, or from at least oneTermamyl-like and at least one fungal alpha-amylase. The Termamyl-likealpha-amylase from which a partial amino acid sequence derives may,e.g., be any of those specific Termamyl-like alpha-amylases referred toherein.

For instance, the parent alpha-amylase may comprise a C-terminal part ofan alpha-amylase derived from a strain of B. licheniformis, and aN-terminal part of an alpha-amylase derived from a strain of B.amyloliquefaciens or from a strain of B. stearothermophilus. Forinstance, the parent alpha-amylase may comprise at least 430 amino acidresidues of the C-terminal part of the B. lichenformis alpha-amylase,and may, e.g., comprise a) an amino acid segment corresponding to the 37N-terminal amino add residues of the B. amyloliquefadens alpha-amylasehaving the amino acid sequence shown in SEQ ID NO: 6 and an amino acidsegment corresponding to the 445 C-terminal amino acid residues of theB. licheniformis alpha-amylase having the amino acid sequence shown inSEQ ID NO: 4, or b) an amino acid segment corresponding to the 68N-terminal amino acid residues of the B. stearothermophilusalpha-amylase having the amino acid sequence shown in SEQ ID NO: 8 andan amino acid segment corresponding to the 415 C-terminal amino acidresidues of the B. licheniformis alpha-amylase having the amino acidsequence shown in SEQ ID NO: 4.

In a preferred embodiment the parent Termamyl-like alpha-amylase is ahybrid Termamyl-like alpha-amylase identical to the Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 4, except that theN-terminal 35 amino acid residues (of the mature protein) is replacedwith the N-terminal 33 amino acid residues of the mature protein of theBacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 6.Said hybrid may further have the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4)referred to as LE174.

Another preferred parent hybrid alpha-amylase is LE429 shown in SEQ IDNO: 2.

The non-Termamyl-like alpha-amylase may, e.g., be a fungalalpha-amylase, a mammalian or a plant alpha-amylase or a bacterialalpha-amylase (different from a Termamyl-like alpha-amylase). Specificexamples of such alpha-amylases include the Aspergillus oryzae TAKAalpha-amylase, the A. niger acid alpha-amylase, the Bacillus subtilisalpha-amylase, the porcine pancreatic alpha-amylase and a barleyalpha-amylase. All of these alpha-amylases have elucidated structures,which are markedly different from the structure of a typicalTermamyl-like alpha-amylase as referred to herein.

The fungal alpha-amylases mentioned above, i.e., derived from A. nigerand A. oryzae, are highly homologous on the amino acid level andgenerally considered to belong to the same family of alpha-amylases. Thefungal alpha-amylase derived from Aspergillus oryzae is commerciallyavailable under the tradename Fungamyl™.

Furthermore, when a particular variant of a Termamyl-like alpha-amylase(variant of the invention) is referred to—in a conventional manner—byreference to modification (e.g., deletion or substitution) of specificamino acid residues in the amino add sequence of a specificTermamyl-like alpha-amylase, it is to be understood that variants ofanother Termamyl-like alpha-amylase modified in the equivalentposition(s) (as determined from the best possible amino acid sequencealignment between the respective amino acid sequences) are encompassedthereby.

A preferred embodiment of a variant of the invention is one derived froma B. lichenformis alpha-amylase (as parent Termamyl-like alpha-amylase),e.g., one of those referred to above, such as the B. licheniformisalpha-amylase having the amino acid sequence shown in SEQ ID NO: 4.

Construction of Variants of the Invention

The construction of the variant of interest may be accomplished bycultivating a microorganism comprising a DNA sequence encoding thevariant under conditions which are conducive for producing the variant.The variant may then subsequently be recovered from the resultingculture broth. This is described in detail further below.

Altered Properties

The following discusses the relationship between mutations, which may bepresent in variants of the invention, and desirable alterations inproperties (relative to those of a parent Termamyl-like alpha-amylase),which may result there from.

In the first aspect the invention relates to a variant of a parentTermamyl-like alpha-amylase, comprising an alteration at one or morepositions selected from the group of:

W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, A111, S168,M197, wherein (a) the alteration(s) are independently

(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position,

(ii) a deletion of the amino acid which occupies the position, or

(iii) a substitution of the amino acid which occupies the position witha different amino acid,

(b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO:4.

In a preferred embodiment the above variants of the invention comprise amutation in a position corresponding to at least one of the followingmutations in the amino acid sequence shown in SEQ ID NO: 4:

-   V54N, A52S, A52S+V54N, T49L, T49+G107A, A52S+V54N+T49L+G107A,    A52S+V54N+T49L, G107A, Q51R, Q51R+A52S, A52N; or-   T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A,    T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A,    T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A;    or-   T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q,    T49K, T49R, A52T, A52L, A52I, A52V, A52M, A52F, A52Y, A52W, V54M,    G107V, G07I, G107L, G107C.

In a preferred embodiment a variant of the invention comprises at leastone mutation in a position corresponding to the following mutations inthe amino acid sequence shown in SEQ ID NO: 4:

-   W13F, L, I, V, Y, A;-   G48A, V, S, T, I, L;-   *48aD or *48aY (i.e., insertion of D or Y);-   T49X;-   *49aX (i.e., insertion of any possible amino acid residue) S50X, in    particular D, Y, L, T, V, I;-   Q51R, K;-   A52X, in particular A52S, N, T, F, L, I, V;-   D53E, Q, Y, I, N, S, T, V, L;-   V54X, in particular V54I, N, W, Y, F, L;-   G57S, A, V, L, I, F, Y, T;-   G107X, in particular G107A, V, S, T, I, L, C;-   G108X, in particular G108A, V, S, T, I, L;-   A111V, I, L;-   S168Y;-   M197X, in particular Y, F, L, I, T, A, G.

In a preferred embodiment a variant of the invention comprises thefollowing mutations corresponding to the following mutations in theamino acid sequence shown in SEQ ID NO: 4:

-   T49X+A52X+V54N/I/L/Y/F/W+G107A, and may further comprise G108A.

In a preferred embodiment a variant of the invention comprises at leastone mutation corresponding to the following mutations in the amino acidsequence shown in SEQ ID NO: 4:

-   T49L+G107A;-   T49I+G107A;-   T49L+G107A+V54I;-   T49I+G107A+V54I;-   A52S+V54N+T49L+G107A;-   A52S+V54I+T49L+G107A;-   A52S+T49L+G107A;-   A52T+T49L+G107A;-   A52S+V54N+T49I+G107A;-   A52S+V54I+T49I+G107A;-   A52S+T49I+G107A;-   T49L+G108A;-   T49I+G108A;-   T49L+G108A+V54I;-   T49I+G108A+V54I.

All of the above-mentioned variants of the invention have alteredproperties (meaning increased or decreased properties), in particular atleast one of the following properties relative to the parentalpha-amylase: reduced ability to cleave a substrate close to thebranching point, improved substrate specificity and/or improved specificactivity, altered substrate binding, altered thermal stability, alteredpH/activity profile, altered pH/stability profile, altered stabilitytowards oxidation, altered Ca²⁺ dependency.

Stability

In the context of the present invention, mutations (including amino acidsubstitutions and/or deletions) of importance with respect to achievingaltered stability, in particular improved stability (i.e., higher orlower), at especially low pH (i.e., pH 4-6) include any of the mutationslisted in the in “Altered properties” section, above and the variantsmentioned right below.

The following variants: Q360A,K; N102A, N326A,L, N190G, N190K; Y262A,K,E(using the BAN, i.e., SEQ ID N: 6, numbering) were also tested for pHstability. A preferred parent alpha-amylase may be BA2 described above.The pH stability was determined as described in the “Materials &Methods” section.

Ca²⁺ Stability

Altered Ca²⁺ stability means the stability of the enzyme under Ca²⁺depletion has been improved, i.e., higher or lower stability. In thecontext of the present invention, mutations (including amino acidsubstitutions) of importance with respect to achieving altered Ca²⁺stability, in particular improved Ca²⁺ stability, i.e., higher or lowerstability, at especially low pH (i.e., pH 4-6) include any of themutations listed in the in “Altered properties” section above.

Specific Activity

In a further aspect of the present invention, important mutations withrespect to obtaining variants exhibiting altered specific activity, inparticular increased or decreased specific activity, especially attemperatures from 60-100° C., preferably 70-95° C., especially 80-90°C., include any of the mutations listed in the in “Altered properties”section above.

The specific activity of LE174 and LE429 was determined to 16,000 NU/mgusing the Phadebas® assay described in the “Materials and Methods”section.

Altered Cleavage Pattern

In the starch liquefaction process it is desirable to use analpha-amylase, which is capable of degrading the starch molecules intolong, branched oligosaccharides, rather than an alpha-amylase, whichgives rise to formation of shorter, branched oligosaccharides (likeconventional Termamyl-like alpha-amylases). Short, branchedoligosaccharides (panose precursors) are not hydrolyzed satisfactorilyby pullulanases, which are used after alpha-amylase treatment in theliquefaction process, or simultaneously with a saccharifyingamyloglucosidase (glucoamylase), or before adding a saccharifyingamyloglucosidase (glucoamylase). Thus, in the presence of panoseprecursors, the product mixture present after the glucoamylase treatmentcontains a significant proportion of short, branched, so-calledlimit-dextrin, viz. the trisaccharide panose. The presence of panoselowers the saccharification yield significantly and is thus undesirable.

It has been reported previously (U.S. Pat. No. 5,234,823) that, whensaccharifying with glucoamylase and pullulanase, the presence ofresidual alpha-amylase activity arising from the liquefaction process,can lead to lower yields of glucose, if the alpha-amylase is notinactivated before the saccharification stage. This inactivation can betypically carried out by adjusting the pH to below 4.7 at 95° C., beforelowering the temperature to 60° C. for saccharification.

The reason for this negative effect on glucose yield is not fullyunderstood, but it is assumed that the liquefying alpha-amylase (forexample Termamyl 120 L from B. licheniformis) generates “limit dextrins”(which are poor substrates for pullulanase), by hydrolysing1,4-alpha-glucosidic linkages close to and on both sides of thebranching points in amylopectin. Hydrolysis of these limit dextrins byglucoamylase leads to a build up of the trisaccharide panose, which isonly slowly hydrolysed by glucoamylase.

The development of a thermostable alpha-amylase, which does not sufferfrom this disadvantage, would be a significant improvement, as noseparate inactivation step would be required.

Thus, the aim of the present invention is to arrive at a mutantalpha-amylase having appropriately modified starch-degradationcharacteristics but retaining the thermostability of the parentTermamyl-like alpha-amylase.

Accordingly, the invention relates to a variant of a Termamyl-likealpha-amylase, which has an improved reduced ability to cleave asubstrate dose to the branching point, and further has improvedsubstrate specificity and/or improved specific activity.

Of particular interest is a variant, which cleaves an amylopectinsubstrate, from the reducing end, more than one glucose unit from thebranching point, preferably more than two or three glucose units fromthe branching point, i.e., at a further distance from the branchingpoint than that obtained by use of a wild type B. licheniformisalpha-amylase.

It may be mentioned here that according to WO 96/23874, variantscomprising at least one of the following mutations are expected toprevent cleavage dose to the branching point:

-   V54L,I,F,Y,W,R,K,H,E,Q;-   D53L,I,F,Y,W;-   Y56W;-   Q333W;-   G57,all possible amino acid residues;-   A52, amino acid residues larger than A, e.g., A52W,Y,L,F,I.

Mutations of particular interest in relation to obtaining variantsaccording to the invention having an improved reduced ability to cleavea substrate close to the branching point, and further has improvedsubstrate specificity and/or improved specific activity includemutations at the following positions in B. licheniformis alpha-amylase,SEQ ID NO: 4: H156, A181, N190, A209, Q264 and I201.

It should be emphazised that not only the Termamyl-like alpha-amylasesmentioned specifically below may be used. Also other commercialTermamyl-like alpha-amylases can be used. An unexhaustive list of suchalpha-amylases is the following:

Alpha-amylases produced by the B. licheniformis strain described in EP0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353and WO 94/18314. Other commercial Termamyl-like B. lichenformisalpha-amylases are Optitherm™ and Takatherm™ (available from Solvay),Maxamyl™ (available from Gist-brocades/Genencor), Spezym AA™ SpezymeDelta AA™ (available from Genencor), and Keistase™ (available fromDaiwa).

All Termamyl-like alpha-amylase may suitably be used as backbone forpreparing variants of the invention.

In a preferred embodiment of the invention the parent Termamyl-likealpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 4 and SEQ ID NO:6. Specifically, the parent hybrid Termamyl-like alpha-amylase may be ahybrid alpha-amylase comprising the 445 C-terminal amino acid residuesof the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37N-terminal amino acid residues of the mature alpha-amylase derived fromB. amyloliquefaciens shown in SEQ ID NO: 6, which may suitably furtherhave the following mutations: H156Y+A181T+N190F+A209V+Q264S (using thenumbering in SEQ ID NO: 4). This hybrid is referred to as LE174. TheLE174 hybrid may be combined with a further mutation I201F to form aparent hybrid Termamyl-like alpha-amylase having the following mutationsH156Y+A181T+N190F+A209V+Q264S+I201F (using SEQ ID NO: 4 for thenumbering). This hybrid variant is shown in SEQ ID NO: 2 and is used inthe examples below, and is referred to as LE429.

Also, LE174 or LE429 (SEQ ID NO: 2) or B. licheniformis alpha-amylaseshown in SEQ ID NO: 4 comprising one or more of the following mutationsmay be used as backbone (using SEQ ID NO: 4 for the numbering of themutations):

-   E119C;-   S130C;-   D124C;-   R127C;-   A52all possible amino acid residues;-   S85all possible amino acid residues;-   N96all possible amino acid residues;-   V129all possible amino acid residues;-   A269all possible amino acid residues;-   A378all possible amino acid residues;-   S148all possible amino acid residues, in particular S148N;-   E211all possible amino acid residues, in particular E211Q;-   N188all possible amino acid residues, in particular N188S, N188P-   M197all possible amino acid residues, in particular M197T, M197A,    M197G, M197I, M197L, M197Y,-   M197F, M197I;-   W138all possible amino acid residues, in particular W138Y;-   D207all possible amino acid residues, in particular D207Y;-   H133all possible amino acid residues, in particular H133Y;-   H205all possible amino acid residues, in particular H205H, H205C,    H205R;-   S187all possible amino acid residues, in particular S187D;-   A210all possible amino acid residues, in particular A210S, A210T;-   H405all possible amino acid residues, in particular H405D;-   K176all possible amino acid residues, in particular K176R;-   F279all possible amino acid residues, in particular F279Y;-   Q298all possible amino acid residues, in particular Q298H;-   G299all possible amino acid residues, in particular G299R;-   L308all possible amino acid residues, in particular L308F;-   T412all possible amino acid residues, in particular T412A;

Further, B. licheniformis alpha-amylase shown in SEQ ID NO: 4 comprisingat least one of the following mutations may be used as backbone:

-   M15all possible amino add residues;-   A33all possible amino acid residues;

When using LE429 (shown in SEQ ID NO: 2) as the backbone (i.e., as theparent Termamyl-like alpha-amylase) by combining LE174 with the mutationI201F (SEQ ID NO: 4 numbering), the mutations/alterations, in particularsubstitutions, deletions and insertions, may according to the inventionbe made in one or more of the following positions to improve the reducedability to cleave a substrate close to the branching point, and toimprove substrate specificity and/or improved specific activity:

-   W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, A111, S168,    M197 using the SEQ ID NO: 4 numbering)    wherein (a) the afteration(s) are independently

(i) an insertion of an amino add downstream of the amino acid whichoccupies the position,

(ii) a deletion of the amino acid which occupies the position, or

(iii) a substitution of the amino acid which occupies the position witha different amino acid,

(b) the variant has alpha-amylase activity and (c) each positioncorresponds to a position of the amino acid sequence of the parentTermamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO:4.

In a preferred embodiment a variant of the invention comprises at leastone mutation in a position corresponding to the following mutations inthe amino acid sequence shown in SEQ ID NO: 4:

-   V54N, A52S, A52S+V54N, T49L, T49+G107A, A52S+V54N+T49L+G107A,    A52S+V54N+T49L, G107A, Q51R, Q51R+A52S, A52N; or-   T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A,    T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A,    T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A;    or-   T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q,    T49K, T49R, A52T, A52L, A52I, A52V, A52M, A52F, A52Y, A52W, V54M,    G107V, G07I, G107L, G107C.

In a preferred embodiment a variant of the invention comprises at leastone mutation in a position corresponding to the following mutations inthe amino acid sequence shown in SEQ ID NO: 4:

-   W13F,L,I,V,Y,A;-   G48A,V,S,T,I,L;-   *48aD or *48aY (i.e., insertion of D or Y);-   T49X;-   *49aX (i.e., insertion of any amino acid residue)-   S50X, in particular D,Y,L,T,V,I;-   Q51R,K;-   A52X, in particular A52S,N,T,F,L,I,V;-   D53E,Q,Y,I,N,S,T,V,L;-   V54X, in particular V54I,N,W,Y,F,L;-   G57S,A,V,L,I,F,Y,T;-   G107X, in particular G107A,V,S,T,I,L,C;-   G108X, in particular G108A,V,S,T,I,L;-   A111V,I,L;-   S168Y;-   M197X, in particular Y,F,L,I,T,A,G.

In a preferred embodiment a variant of the invention comprises at leastone mutation in a position corresponding to the following mutations inthe amino acid sequence shown in SEQ ID NO: 4:

-   -   T49X+A52X+V54N/I/L/Y/F/W+G107A, and may further comprise G108A.

In a preferred embodiment a variant of the invention comprises at leastone mutation in a position corresponding to the following mutations inthe amino acid sequence shown in SEQ ID NO: 4:

-   T49L+G107A;-   T49I+G107A;-   T49L+G107A+V54I;-   T49I+G107A+V54I;-   A52S+V54N+T49L+G107A;-   A52S+V54I+T49L+G107A;-   A52S+T49L+G107A;-   A52T+T49L+G107A;-   A52S+V54N+T49I+G107A;-   A52S+V54I+T49I+G107A;-   A52S+T49I+G107A;-   T49L+G108A;-   T49I+G108A;-   T49L+G108A+V54I;-   T49I+G108A+V54I.    General Mutations in Variants of the Invention

It may be preferred that a variant of the invention comprises one ormore modifications in addition to those outlined above. Thus, it may beadvantageous that one or more proline residues present in the part ofthe alpha-amylase variant which is modified is/are replaced with anon-proline residue which may be any of the possible, naturallyoccurring non-proline residues, and which preferably is an alanine,glycine, serine, threonine, valine or leucine.

Analogously, it may be preferred that one or more cysteine residuespresent among the amino acid residues with which the parentalpha-amylase is modified is/are replaced with a non-cysteine residuesuch as serine, alanine, threonine, glycine, valine or leucine.

Furthermore, a variant of the invention may—either as the onlymodification or in combination with any of the above outlinedmodifications—be modified so that one or more Asp and/or Glu present inan amino acid fragment corresponding to the amino acid fragment 185-209of SEQ ID NO. 4 is replaced by an Asn and/or Gln, respectively. Also ofinterest is the replacement, in the Termamyl-like alpha-amylase, of oneor more of the Lys residues present in an amino acid fragmentcorresponding to the amino acid fragment 185-209 of SEQ ID NO: 4 by anArg.

It will be understood that the present invention encompasses variantsincorporating two or more of the above outlined modifications.

Furthermore, it may be advantageous to introduce point-mutations in anyof the variants described herein.

Methods for Preparing Alpha-amylase Variants

Several methods for introducing mutations into genes are known in theart. After a brief discussion of the cloning of alpha-amylase-encodingDNA sequences, methods for generating mutations at specific sites withinthe alpha-amylase-encoding sequence will be discussed.

Cloning a DNA Sequence Encoding an Alpha-amylase

The DNA sequence encoding a parent alpha-amylase may be isolated fromany cell or microorganism producing the alpha-amylase in question, usingvarious methods well known in the art. First, a genomic DNA and/or cDNAlibrary should be constructed using chromosomal DNA or messenger RNAfrom the organism that produces the alpha-amylase to be studied. Then,if the amino acid sequence of the alpha-amylase is known, homologous,labelled oligonucleotide probes may be synthesized and used to identifyalpha-amylase-encoding clones from a genomic library prepared from theorganism in question. Alternatively, a labelled oligonucleotide probecontaining sequences homologous to a known alpha-amylase gene could beused as a probe to identify alpha-amylase-encoding clones, usinghybridization and washing conditions of lower stringency.

Yet another method for identifying alpha-amylase-encoding clones wouldinvolve inserting fragments of genomic DNA into an expression vector,such as a plasmid, transforming alpha-amylase-negative bacteria with theresulting genomic DNA library, and then plating the transformed bacteriaonto agar containing a substrate for alpha-amylase, thereby allowingclones expressing the alpha-amylase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be preparedsynthetically by established standard methods, e.g., thephosphoroamidite method described by S. L. Beaucage and M. H. Caruthers(1981) or the method described by Matthes et al. (1984). In thephosphoroamidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate, the fragments corresponding to various parts of the entireDNA sequence), in accordance with standard techniques. The DNA sequencemay also be prepared by polymerase chain reaction (PCR) using specificprimers, for instance as described in U.S. Pat. No. 4,683,202 or R. K.Saiki et al. (1988).

Site-Directed Mutagenesis

Once an alpha-amylase-encoding DNA sequence has been isolated, anddesirable sites for mutation identified, mutations may be introducedusing synthetic oligonudeotides. These oligonucleotides containnucleotide sequences flanking the desired mutation sites; mutantnucleotides are inserted during oligonucleotide synthesis. In a specificmethod, a single-stranded gap of DNA, bridging thealpha-amylase-encoding sequence, is created in a vector carrying thealpha-amylase gene. Then the synthetic nudeotide, bearing the desiredmutation, is annealed to a homologous portion of the single-strandedDNA. The remaining gap is then filled in with DNA polymerase I (Klenowfragment) and the construct is ligated using T4 ligase. A specificexample of this method is described in Morinaga et al. (1984). U.S. Pat.No. 4,760,025 disclose the introduction of oligonudeotides encodingmultiple mutations by performing minor alterations of the cassette.However, an even greater variety of mutations can be introduced at anyone time by the Morinaga method, because a multitude ofoligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase-encoding DNAsequences is described in Nelson and Long (1989). It involves the 3-stepgeneration of a PCR fragment containing the desired mutation introducedby using a chemically synthesized DNA strand as one of the primers inthe PCR reactions. From the PCR-generated fragment, a DNA fragmentcarrying the mutation may be isolated by cleavage with restrictionendonucleases and reinserted into an expression plasmid.

Random Mutagenesis

Random mutagenesis is suitably performed either as localised orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene.

The random mutagenesis of a DNA sequence encoding a parent alpha-amylasemay be conveniently performed by use of any method known in the art.

In relation to the above, a further aspect of the present inventionrelates to a method for generating a variant of a parent alpha-amylase,e.g., wherein the variant exhibits a reduced capability of cleaving anoligo-saccharide substrate close to the branching point, and furtherexhibits improved substrate specificity and/or improved specificactivity relative to the parent, the method:

-   -   (a) subjecting a DNA sequence encoding the parent alpha-amylase        to random mutagenesis,    -   (b) expressing the mutated DNA sequence obtained in step (a) in        a host cell, and    -   (c) screening for host cells expressing an alpha-amylase variant        which has an altered property (i.e., thermal stability) relative        to the parent alpha-amylase.

Step (a) of the above method of the invention is preferably performedusing doped primers. For instance, the random mutagenesis may beperformed by use of a suitable physical or chemical mutagenizing agent,by use of a suitable oligonucleotide, or by subjecting the DNA sequenceto PCR generated mutagenesis. Furthermore, the random mutagenesis may beperformed by use of any combination of these mutagenizing agents. Themutagenizing agent may, e.g., be one, which induces transitions,transversions, inversions, scrambling, deletions, and/or insertions.Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) ir-radiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues. When such agents are used, themutagenesis is typically performed by incubating the DNA sequenceencoding the parent enzyme to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions for themutagenesis to take place, and selecting for mutated DNA having thedesired properties. When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions, which are to be changed. The doping or spiking may bedone so that codons for unwanted amino acids are avoided. The doped orspiked oligonucleotide can be incorporated into the DNA encoding thealpha-amylase enzyme by any published technique, using e.g., PCR, LCR orany DNA polymerase and ligase as deemed appropriate. Preferably, thedoping is carried out using “constant random doping”, in which thepercentage of wild type and mutation in each position is predefined.Furthermore, the doping may be directed toward a preference for theintroduction of certain nudeotides, and thereby a preference for theintroduction of one or more specific amino acid residues. The doping maybe made, e.g., so as to allow for the introduction of 90% wild type and10% mutations in each position. An additional consideration in thechoice of a doping scheme is based on genetic as well asprotein-structural constraints. The doping scheme may be made by usingthe DOPE program, which, inter alia, ensures that introduction of stopcodons is avoided. When PCR-generated mutagenesis is used, either achemically treated or non-treated gene encoding a parent alpha-amylaseis subjected to PCR under conditions that increase the mis-incorporationof nucleotides (Deshler 1992; Leung et al., Technique, Vol.1, 1989, pp.11-15). A mutator strain of E. coli (Fowleret al., Molec. Gen. Genet.,133, 1974, pp. 179-191), S. cereviseae or any other microbial organismmay be used for the random mutagenesis of the DNA encoding thealpha-amylase by, e.g., transforming a plasmid containing the parentglycosylase into the mutator strain, growing the mutator strain with theplasmid and isolating the mutated plasmid from the mutator strain. Themutated plasmid may be subsequently transformed into the expressionorganism. The DNA sequence to be mutagenized may be conveniently presentin a genomic or cDNA library prepared from an organism expressing theparent alpha-amylase. Alternatively, the DNA sequence may be present ona suitable vector such as a plasmid or a bacteriophage, which as suchmay be incubated with or otherwise exposed to the mutagenising agent.The DNA to be mutagenized may also be present in a host cell either bybeing integrated in the genome of said cell or by being present on avector harboured in the cell. Finally, the DNA to be mutagenized may bein isolated form. It will be understood that the DNA sequence to besubjected to random mutagenesis is preferably a cDNA or a genomic DNAsequence. In some cases it may be convenient to amplify the mutated DNAsequence prior to performing the expression step b) or the screeningstep c). Such amplification may be performed in accordance with methodsknown in the art, the presently preferred method being PCR-generatedamplification using oligonucleotide primers prepared on the basis of theDNA or amino acid sequence of the parent enzyme. Subsequent to theincubation with or exposure to the mutagenising agent, the mutated DNAis expressed by culturing a suitable host cell carrying the DNA sequenceunder conditions allowing expression to take place. The host cell usedfor this purpose may be one which has been transformed with the mutatedDNA sequence, optionally present on a vector, or one which was carriedthe DNA sequence encoding the parent enzyme during the mutagenesistreatment. Examples of suitable host cells are the following: grampositive bacteria such as Bacillus subtilis, Bacillus licheniformis,Bacillus lentus, Bacillus brevis, Bacillus stearotherrnophilus, Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacilluscirculans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis,Streptomyces lividans or Streptomyces murinus; and gram-negativebacteria such as E. coli. The mutated DNA sequence may further comprisea DNA sequence encoding functions permitting expression of the mutatedDNA sequence.

Localised Random Mutagenesis

The random mutagenesis may be advantageously localised to a part of theparent alpha-amylase in question. This may, e.g., be advantageous whencertain regions of the enzyme have been identified to be of particularimportance for a given property of the enzyme, and when modified areexpected to result in a variant having improved properties. Such regionsmay normally be identified when the tertiary structure of the parentenzyme has been elucidated and related to the function of the enzyme.

The localised, or region-specific, random mutagenesis is convenientlyperformed by use of PCR generated mutagenesis techniques as describedabove or any other suitable technique known in the art. Alternatively,the DNA sequence encoding the part of the DNA sequence to be modifiedmay be isolated, e.g., by insertion into a suitable vector, and saidpart may be subsequently subjected to mutagenesis by use of any of themutagenesis methods discussed above.

Altemative Methods of Providing Alpha-amylase Variants

Alternative methods for providing variants of the invention includegene-shuffling method known in the art including the methods e.g.,described in WO 95/22625 (from Affymax Technologies N.V.) and WO96/00343 (from Novo Nordisk A/S).

Expression of Alpha-amylase Variants

According to the invention, a DNA sequence encoding the variant producedby methods described above, or by any alternative methods known in theart, can be expressed, in enzyme form, using an expression vector whichtypically includes control sequences encoding a promoter, operator,ribosome binding site, translation initiation signal, and, optionally, arepressor gene or various activator genes.

The recombinant expression vector carrying the DNA sequence encoding analpha-amylase variant of the invention may be any vector, which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e., a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid, a bacteriophage or an extrachromosomal element, minichromosomeor an artificial chromosome. Alternatively, the vector may be one which,when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

In the vector, the DNA sequence should be operably connected to asuitable promoter sequence. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell. Examples of suitable promoters for directing thetranscription of the DNA sequence encoding an alpha-amylase variant ofthe invention, especially in a bacterial host, are the promoter of thelac operon of E. coli, the Streptomyces coelicolor agarase gene dagApromoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the promoters of the Bacillus stearothermophilus maltogenicamylase gene (amyM), the promoters of the Bacillus amyloliquefaciensalpha-amylase (amyQ), the promoters of the Bacillus subfilis xylA andxylB genes etc. For transcription in a fungal host, examples of usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralalpha-amylase, A. niger acid stable alpha-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A.oryzae triose phosphate isomerase or A. nidulans acetamidase.

The expression vector of the invention may also comprise a suitabletranscription terminator and, in eukaryotes, polyadenylation sequencesoperably connected to the DNA sequence encoding the alpha-amylasevariant of the invention. Termination and polyadenylation sequences maysuitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. Examples of such sequences arethe origins of replication of plasmids pUC19, pACYC177, pUB110, pE194,pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, such as the dalgenes from B. subtilis or B. licheniformis, or one which confersantibiotic resistance such as ampicillin, kanamycin, chloramphenicol ortetracyclin resistance. Furthermore, the vector may comprise Aspergillusselection markers such as amdS, argB, niaD and sC, a marker giving riseto hygromycin resistance, or the selection may be accomplished byco-transformation, e.g., as described in WO 91/17243.

While intracellular expression may be advantageous in some respects,e.g., when using certain bacteria as host cells, it is generallypreferred that the expression is extracellular. In general, the Bacillusalpha-amylases mentioned herein comprise a pre-region permittingsecretion of the expressed protease into the culture medium. Ifdesirable, this pre-region may be replaced by a different preregion orsignal sequence, conveniently accomplished by substitution of the DNAsequences encoding the respective preregions.

The procedures used to ligate the DNA construct of the inventionencoding an alpha-amylase variant, the promoter, terminator and otherelements, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989).

The cell of the invention, either comprising a DNA construct or anexpression vector of the invention as defined above, is advantageouslyused as a host cell in the recombinant production of an alpha-amylasevariant of the invention. The cell may be transformed with the DNAconstruct of the invention encoding the variant, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal or an insect, but is preferably a microbial cell, e.g., abacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are gram-positive bacteria such asBacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillusbrevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacilluslautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyceslividans or Streptomyces murinus, or gramnegative bacteria such as E.coli. The transformation of the bacteria may, for instance, be effectedby protoplast transformation or by using competent cells in a mannerknown per se.

The yeast organism may favourably be selected from a species ofSaccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae.The filamentous fungus may advantageously belong to a species ofAspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cellsmay be transformed by a process involving protoplast formation andtransformation of the protoplasts followed by regeneration of the cellwall in a manner known per se. A suitable procedure for transformationof Aspergillus host cells is described in EP 238 023.

In yet a further aspect, the present invention relates to a method ofproducing an alpha-amylase variant of the invention, which methodcomprises cultivating a host cell as described above under conditionsconducive to the production of the variant and recovering the variantfrom the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional mediumsuitable for growing the host cell in question and obtaining expressionof the alpha-amylase variant of the invention. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g., as described in catalogues of the American TypeCulture Collection).

The alpha-amylase variant secreted from the host cells may convenientlybe recovered from the culture medium by well-known procedures, includingseparating the cells from the medium by centrifugation or filtration,and precipitating proteinaceous components of the medium by means of asalt such as ammonium sulphate, followed by the use of chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Industrial Applications

The alpha-amylase variants of this invention possess valuable propertiesallowing for a variety of industrial applications. In particular, enzymevariants of the invention are applicable as a component in washing,dishwashing and hard surface cleaning detergent compositions. Numerousvariants are particularly useful in the production of sweeteners andethanol, e.g., fuel, drinking or industrial ethanol, from starch, and/orfor textile desizing. Conditions for conventional starch-conversionprocesses, including starch liquefaction and/or saccharificationprocesses, are described in, e.g., U.S. Pat. No. 3,912,590 and in EPpatent publications Nos. 252 730 and 63 909.

Production of Sweeteners from Starch

A “traditional” process for conversion of starch to fructose syrupsnormally consists of three consecutive enzymatic processes, viz. aliquefaction process followed by a saccharification process and anisomerization process. During the liquefaction process, starch isdegraded to dextrins by an alpha-amylase (e.g., Termamyl™) at pH valuesbetween 5.5 and 6.2 and at temperatures of 95-160° C. for a period ofapprox. 2 hours. In order to ensure optimal enzyme stability under theseconditions, 1 mM of calcium is added (40 ppm free calcium ions).

After the liquefaction process the dextrins are converted into dextroseby addition of a glucoamylase (e.g., AMG™) and a debranching enzyme,such as an isoamylase or a pullulanase (e.g., Promozyme™). Before thisstep the pH is reduced to a value below 4.5, maintaining the hightemperature (above 95° C.), and the liquefying alpha-amylase activity isdenatured. The temperature is lowered to 60° C., and glucoamylase anddebranching enzyme are added. The saccharification process proceeds for24-72 hours.

After the saccharification process the pH is increased to a value in therange of 6-8, preferably pH 7.5, and the calcium is removed by ionexchange. The dextrose syrup is then converted into high fructose syrupusing, e.g., an immmobilized glucoseisomerase (such as Sweetzyme™).

At least one enzymatic improvement of this process could be envisaged:Reduction of the calcium dependency of the liquefying alpha-amylase.Addition of free calcium is required to ensure adequately high stabilityof the alpha-amylase, but free calcium strongly inhibits the activity ofthe glucoseisomerase and needs to be removed, by means of an expensiveunit operation, to an extent, which reduces the level of free calcium tobelow 3-5 ppm. Cost savings could be obtained if such an operation couldbe avoided and the liquefaction process could be performed withoutaddition of free calcium ions.

To achieve that, a less calcium-dependent Termamyl-like alpha-amylasewhich is stable and highly active at low concentrations of free calcium(<40 ppm) is required. Such a Termamyl-like alpha-amylase should have apH optimum at a pH in the range of 4.5-6.5, preferably in the range of4.5-5.5.

The invention also relates to a composition comprising a mixture of oneor more variants of the invention derived from (as the parentTermamyl-like alpha-amylase) the B. stearothermophilus alpha-amylasehaving the sequence shown in SEQ ID NO: 8 and a Termamyl-likealpha-amylase derived from the B. licheniformis alpha-amylase having thesequence shown in SEQ ID NO: 4.

Further, the invention also relates to a composition comprising amixture of one or more variants according the invention derived from (asthe parent Termamyl-like alpha-amylase) the B. stearothermophilusalpha-amylase having the sequence shown in SEQ ID NO: 8 and a hybridalpha-amylase comprising a part of the B. amyloliquefaciensalpha-amylase shown in SEQ ID NO: 6 and a part of the B. licheniformisalpha-amylase shown in SEQ ID NO: 4. The latter mentioned hybridTermamyl-like alpha-amylase comprises the 445 C-terminal amino acidresidues of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 andthe 37 N-terminal amino acid residues of the alpha-amylase derived fromB. amyloliquefaciens shown in SEQ ID NO: 6. Said latter mentioned hybridalpha-amylase may suitably comprise the following mutations:H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4)Preferably, said latter mentioned hybrid alpha-amylase may suitablycomprise the following mutations: H156Y+A181T+N190F+A209V+Q264S+I201F(using the SEQ ID NO: 4 numbering). In the examples below saidlast-mentioned parent hybrid Termamyl-like alpha-amylase referred to asLE429 (shown in SEQ ID NO: 2) is used for preparing variants of theinvention, which variants may be used in compositions of the invention.

An alpha-amylase variant of the invention or a composition of theinvention may in an aspect of the invention be used for starchliquefaction, in detergent composition, such as laundry, dish washcompositions and hard surface cleaning, ethanol production, such asfuel, drinking and industrial ethanol production, desizing of textile,fabric and garments.

Materials and Methods

Enzymes

LE174: Hybrid Alpha-amylase Variant

LE174 is a hybrid Termamyl-like alpha-amylase being identical to theTermamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shownin SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (ofthe mature protein) has been replaced by the N-terminal 33 residues ofBAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylaseshown in SEQ ID NO: 6, which further have following mutations:

H156Y+A181T+N190F+A209V+Q264S (SEQ ID NO: 4).

LE429 Hybrid Alpha-amylase Variant

LE429 is a hybrid Termamyl-like alpha-amylase being identical to theTermamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shownin SEQ ID NO: 4, except that the N-terminal 35 amino acid residues (ofthe mature protein) has been replaced by the N-terminal 33 residues ofBAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylaseshown in SEQ ID NO: 6, which further have following mutations:

H156Y+A181T+N190F+A209V+Q264S+I201F (SEQ ID NO: 4). LE429 is shown asSEQ ID NO: 2 and was constructed by SOE-PCR (Higuchi et al. 1988,Nucleic Acids Research 16:7351). Dextrozyme™ E: a balanced mixture ofglucoamylase (AMG) and pullulanase obtainable from selected strains ofAspergillus niger and Bacillus deramificans (available from Novo NordiskA/S)

Fermentation and Purification of Alpha-amylase Variants

A B. subtilis strain harbouring the relevant expression plasmid isstreaked on an LB-agar plate with 10 micro g/ml kanamycin from −80° C.stock, and grown overnight at 37° C. The colonies are transferred to 100ml BPX media supplemented with 10 micro g/ml kanamycin in a 500 mlshaking flask.

Composition of BPX medium:

Potato starch 100 g/l Barley flour 50 g/l BAN 5000 SKB 0.1 g/l Sodiumcaseinate 10 g/l Soy Bean Meal 20 g/l Na₂HPO₄, 12 H₂O 9 g/l Pluronic ™0.1 g/l

The culture is shaken at 37° C. at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth bycentrifugation at 4500 rpm in 20-25 minutes. Afterwards the supernatantis filtered to obtain a completely clear solution. The filtrate isconcentrated and washed on an UF-filter (10000 cut off membrane) and thebuffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied ona S-sepharose F.F. and elution is carried out by step elution with 0.2 MNaCl in the same buffer. The eluate is dialysed against 10 mM Tris, pH9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradientfrom 0-0.3M NaCl over 6 column volumes. The fractions that contain theactivity (measured by the Phadebas assay) are pooled, pH was adjusted topH 7.5 and remaining color was removed by a treatment with 0.5% W/vol.active coal in 5 minutes.

Activity Determination—(KNU)

One Kilo alpha-amylase Unit (1 KNU) is the amount of enzyme which breaksdown 5.26 g starch (Merck, Amylum Solubile, Erg. B 6, Batch 9947275) perhour in Novo Nordisk's standard method for determination ofalpha-amylase based upon the following condition:

Substrate soluble starch Calcium content in solvent 0.0043 M Reactiontime 7-20 minutes Temperature 37° C. pH 5.6

Detailed description of Novo Nordisk's analytical method (AF 9) isavailable on request.

Assay for Alpha-Amylase Activity

Alpha-Amylase activity is determined by a method employing Phadebas®tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, suppliedby Pharmacia Diagnostic) contain a cross-linked insoluble blue-colouredstarch polymer, which has been mixed with bovine serum albumin and abuffer substance and tabletted.

For every single measurement one tablet is suspended in a tubecontaining 5 ml 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mMphosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂, pH adjusted to thevalue of interest with NaOH). The test is performed in a water bath atthe temperature of interest. The alpha-amylase to be tested is dilutedin x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylasesolution is added to the 5 ml 50 mM Britton-Robinson buffer. The starchis hydrolysed by the alpha-amylase giving soluble blue fragments. Theabsorbance of the resulting blue solution, measuredspectrophotometrically at 620 nm, is a function of the alpha-amylaseactivity.

It is important that the measured 620 nm absorbance after 10 or 15minutes of incubation (testing time) is in the range of 0.2 to 2.0absorbance units at 620 nm. In this absorbance range there is linearitybetween activity and absorbance (Lambert-Beer law). The dilution of theenzyme must therefore be adjusted to fit this criterion. Under aspecified set of conditions (temp., pH, reaction time, bufferconditions) 1 mg of a given alpha-amylase will hydrolyse a certainamount of substrate and a blue colour will be produced. The colourintensity is measured at 620 nm. The measured absorbance is directlyproportional to the specific activity (activity/mg of pure alpha-amylaseprotein) of the alpha-amylase in question under the given set ofconditions.

Determining Specific Activity

The specific activity is determined using the Phadebas assay (Pharmacia)as activity/mg enzyme.

Measuring the pH Activity Profile (pH Stability)

The variant is stored in 20 mM TRIS ph 7.5, 0.1 mM, CaCl₂ and tested at30? C, 50 mM Brifton-Robinson, 0.1 mM CaCl₂. The pH activity is measuredat pH 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 9.5, 9.5, 10, and 10.5, usingthe Phadebas assay described above.

Determination of AGU Activity and as AGU/mg

One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute at 37° C. and pH 4.3. Adetailed description of the analytical method (AEL-SM-0131) is availableon request from Novo Nordisk.

The activity is determined as AGU/ml by a method modified after(AEL-SM-0131) using the Glucose GOD-Perid kit from Boehringer Mannheim,124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml.

375 microL substrate (1% maltose in 50 mM Sodium acetate, pH 4.3) isincubated 5 minutes at 37° C. 25 microL enzyme diluted in sodium acetateis added. The reaction is stopped after 10 minutes by adding 100 microL0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and200 microL GOD-Perid solution is added. After 30 minutes at roomtemperature, the absorbance is measured at 650 nm and the activitycalculated in AGU/ml from the AMG-standard.

The specific activity in AGU/mg is then calculated from the activity(AGU/ml) divided with the protein concentration (mg/ml).

EXAMPLES Example 1

Construction of Termamyl Variants in Accordance with the Invention

Termamyl (B. licheniformis alpha-amylase SEQ ID NO: 4) is expressed inB. subtilis from a plasmid denoted pDN1528. This plasmid contains thecomplete gene encoding Termamyl, amyl, the expression of which isdirected by its own promoter. Further, the plasmid contains the originof replication, ori, from plasmid pUB 110 and the cat gene from plasmidpC 194 conferring resistance towards chloramphenicol. pDN1528 is shownin FIG. 9 of WO 96/23874. A specific mutagenesis vector containing amajor part of the coding region of SEQ ID NO: 3 was prepared. Theimportant features of this vector, denoted pJeEN1, include an origin ofreplication derived from the pUC plasmids, the cat gene conferringresistance towards chloramphenicol, and a frameshift-containing versionof the bla gene, the wild type of which normally confers resistancetowards ampicillin (amp^(R) phenotype). This mutated version results inan amp^(S) phenotype. The plasmid pJeEN1 is shown in FIG. 10 of WO96/23874, and the E. coli origin of replication, ori, bla, cat, the5′-truncated version of the Termamyl amylase gene, and selectedrestriction sites are indicated on the plasmid.

Mutations are introduced in amyL by the method described by Deng andNickoloff (1992, Anal. Biochem. 200, pp. 81-88) except that plasmidswith the “selection primer” (primer #6616; see below) incorporated areselected based on the amp^(R) phenotype of transformed E. coli cellsharboring a plasmid with a repaired bla gene, instead of employing theselection by restriction enzyme digestion outlined by Deng andNickoloff. Chemicals and enzymes used for the mutagenesis were obtainedfrom the ChameleonÔ mutagenesis kit from Stratagene (cataloguenumber200509).

After verification of the DNA sequence in variant plasmids, thetruncated gene, containing the desired alteration, is subdoned intopDN1528 as a Pstl-EcoRI fragment and transformed into the protease- andamylase-depleted Bacillus subtilis strain SHA273 (described inWO92/11357 and WO95/10603) in order to express the variant enzyme.

The Termamyl variant V54W was constructed by the use of the followingmutagenesis primer (written 5′ to 3′, left to right):

-   PG GTC GTA GGC ACC GTA GCC CCA ATC CGC TTG (SEQ ID NO: 9)

The Termamyl variant A52W+V54W was constructed by the use of thefollowing mutagenesis primer (written 5′ to 3′, left to right):

-   PG GTC GTA GGC ACC GTA GCC CCA ATC CCA TTG GCT CG (SEQ ID NO: 10)

Primer #6616 (written 5′ to 3′, left to right; P denotes a 5′phosphate):

-   P CTG TGA CTG GTG AGT ACT CAA CCA AGT C (SEQ ID NO: 11)

The Termamyl variant V54E was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PGG TCG TAG GCA CCG TAG CCC TCA TCC GCT TG (SEQ ID NO: 12)

The Termamyl variant V54M was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PGG TCG TAG GCA CCG TAG CCC ATA TCC GCT TG (SEQ ID NO: 13)

The Termamyl variant V541 was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PGG TCG TAG GCA CCG TAG CCA ATA TCC GCT TG (SEQ ID NO: 14)

The Termamyl variants Y290E and Y290K were constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

-   PGC AGC ATG GAA CTG CTY ATG AAG AGG CAC GTC AAA C (SEQ ID NO:15)

Y represents an equal mixture of C and T. The presence of a codonencoding either Glutamate or Lysine in position 290 was verified by DNAsequencing.

The Termamyl variant N190F was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PCA TAG TTG CCG AAT TCA TTG GAA ACT TCC C (SEQ ID NO: 16)

The Termamyl variant N188P+N190F was constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

-   PCA TAG TTG CCG AAT TCA GGG GAA ACT TCC CAA TC (SEQ ID NO: 17)

The Termamyl variant H140K+H142D was constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

-   PCC GCG CCC CGG GAA ATC AAA TTT TGT CCA GGC TTT AAT TAG (SEQ ID NO:    18)

The Termamyl variant H156Y was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PCA AAA TGG TAC CAA TAC CAC TTA AAA TCG CTG (SEQ ID NO: 19)

The Termamyl variant A181T was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PCT TCC CAA TCC CAA GTC TTC CCT TGA AAC (SEQ ID NO: 20)

The Termamyl variant A209V was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PCTT AAT TTC TGC TAC GAC GTC AGG ATG GTC ATA ATC (SEQ ID NO: 21)

The Termamyl variant Q264S was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PCG CCC AAG TCA TTC GAC CAG TAC TCA GCT ACC GTA AAC (SEQ ID NO: 22)

The Termamyl variant S187D was constructed by the use of the followingmutagenesis primer (written 5′-3′, left to right):

-   PGC CGT TTT CAT TGT CGA CTT CCC AAT CCC (SEQ ID NO: 23)

The Termamyl variant DELTA(K370-G371-D372) (i.e., deleted of amino acidresidues nos. 370, 371 and 372) was constructed by the use of thefollowing mutagenesis primer (written 5′-3′, left to right):

-   PGG AAT TTC GCG CTG ACT AGT CCC GTA CAT ATC CCC (SEQ ID NO: 24)

The Termamyl variant DELTA(D372-S373-Q374) was constructed by the use ofthe following mutagenesis primer (written 5′-3′, left to right):

-   PGG CAG GAA TTT CGC GAC CTT TCG TCC CGT ACA TAT C (SEQ ID NO: 25)

The Termamyl variants A181T and A209V were combined to A181T+A209V bydigesting the A181T containing pDN1528-like plasmid (i.e., pDN1528containing within amyL the mutation resulting in the A181T alteration)and the A209V-containing pDN1528-like plasmid (i.e., pDN1528 containingwithin amyL the mutation resulting in the A209V alteration) withrestriction enzyme Clal which cuts the pDN1528-like plasmids twiceresulting in a fragment of 1116 bp and the vector-part (i.e. containsthe plasmid origin of replication) of 3850 bp. The fragment containingthe A209V mutation and the vector part containing the A181T mutationwere purified by QIAquick gel extraction kit (purchased from QIAGEN)after separation on an agarose gel. The fragment and the vector wereligated and transformed into the protease and amylase depleted Bacillussubtilis strain referred to above. Plasmid from amy+ (clearing zones onstarch containing agar-plates) and chloramphenicol resistanttransformants were analysed for the presence of both mutations on theplasmid.

In a similar way as described above, H156Y and A209V were combinedutilizing restriction endonucleases Acc651 and EcoRI, givingH156Y+A209V.

H156Y+A209V and A181T+A209V were combined into H156Y+A181T+A209V by theuse of restriction endonucleases Acc651 and HindIII.

The 35 N-terminal residues of the mature part of Termamyl variantH156Y+A181T+A209V were substituted by the 33 N-terminal residues of theB. amyloliquefaciens alpha-amylase (SEQ ID NO: 4) (which in the presentcontext is termed BAN) by a SOE-PCR approach (Higuchi et al. 1988,Nucleic Acids Research 16:7351) as follows:

Primer 19364 (sequence 5′-3′): CCT CAT TCT GCA GCA GCA GCC GTA AAT GGCACG CTG (SEQ ID NO:26) Primer 19362: CCA GAC GGC AGT AAT ACC GAT ATC CGATAA ATG TTC CG (SEQ ID NO:27) Primer 19363: CGG ATA TCG GTA TTA CTG CCGTCT GGA TTC (SEQ ID NO:28) Primer 1C: CTC GTC CCA ATC GGT TCC GTC (SEQID NO:29)

A standard PCR, polymerase chain reaction, was carried out using the Pwothermostable polymerase from Boehringer Mannheim according to themanufacturer's instructions and the temperature cyclus: 5 minutes at 94°C., 25 cycles of (94° C. for 30 seconds, 50° C. for 45 seconds, 72° C.for 1 minute), 72° C. for 10 minutes.

An approximately 130 bp fragment was amplified in a first PCR denotedPCR1 with primers 19364 and 19362 on a DNA fragment containing the geneencoding the B. amyloliquefaciens alpha-amylase.

An approximately 400 bp fragment was amplified in another PCR denotedPCR2 with primers 19363 and 1C on template pDN1528.

PCR1 and PCR2 were purified from an agarose gel and used as templates inPCR3 with primers 19364 and 1C, which resulted in a fragment ofapproximately 520 bp. This fragment thus contains one part of DNAencoding the N-terminus from BAN fused to a part of DNA encodingTermamyl from the 35th amino acid.

The 520 bp fragment was subcloned into a pDN1528-like plasmid(containing the gene encoding Termamyl variant H156Y+A181T+A209V) bydigestion with restriction endonucleases Pstl and SacIl, ligation andtransformation of the B. subtilis strain as previously described. TheDNA sequence between restriction sites Pstl and SacIl was verified byDNA sequencing in extracted plasmids from amy+ and chloramphenicolresistant transformants.

The final construct containing the correct N-terminus from BAN andH156Y+A181T+A209V was denoted BAN(1-35)+H156Y+A181T+A209V.

N190F was combined with BAN(1-35)+H156Y+A181T+A209V givingBAN(1-35)+H156Y+A181T+N190F+A209V by carrying out mutagenesis asdescribed above except that the sequence of amyL in pJeEN1 wassubstituted by the DNA sequence encoding Termamyl variantBAN(1-35)+H156Y+A181T+A209V

Q264S was combined with BAN(1-35)+H156Y+A181T+A209V givingBAN(1-35)+H156Y+A181T+A209V+Q264S by carrying out mutagenesis asdescribed above except that the sequence of amyL in pJeEN wassubstituted by the the DNA sequence encoding Termamyl variantBAN(1-35)+H156Y+A181T+A209V

BAN(1-35)+H156Y+A181T+A209V+Q264S and BAN(1-35)+H156Y+A181T+N190F+A209Vwere combined into BAN(1-35)+H156Y+A181T+N190F+A209V+Q264S utilizingrestriction endonucleases BsaHI (BsaHI site was introduced close to theA209V mutation) and Pstl. I201F was combined withBAN(1-35)+H156Y+A181T+N190F+A209V+Q264S givingBAN(1-35)+H156Y+A181T+N190F+A209V+Q264S+I201F (SEQ ID NO: 2) by carryingout mutagenesis as described above. The mutagenesis primer AM100 wasused, introduced the I201 F substitution and removed simultaneously aCla I restriction site, which facilitates easy pin-pointing of mutants.

primer AM100:

-   5′GATGTATGCCGACTTCGATTATGACC 3′ (SEQ ID NO: 30

Example 2

Construction of Termamyl-like alpha-amylase Variants with an AlteredCleavage Pattern According to the Invention

The variant of the thermostable B. licheniformis alpha-amylaseconsisting comprising the 445 C-terminal amino acid residues of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 and the 37 N-terminalamino acid residues of the alpha-amylase derived from B.amyloliquefaciens shown in SEQ ID NO: 6, and further comprising thefollowing mutations: H156Y+A181T+N190F+A209V+Q264S+I201F (theconstruction of this variant is described in Example 1, and the aminoacid sequence shown in SEQ ID NO: 2) has a reduced capability ofcleaving an substrate close to the branching point.

In an attempt to further improve the reduced capability of cleaving ansubstrate close to the branching point of said alpha-amylase variantsite directed mutagenesis was carried out using the Mega-primer methodas described by Sarkar and Sommer, 1990 (BioTechniques 8: 404-407):

Construction of LE313: RAN/TermamylHybrid+H156Y+A181T+N190F+A209V+Q264S+V54N

Gene specific primer 27274 and mutagenic primer AM115 are used toamplify by PCR an approximately 440 bp DNA fragment from a pDN1528-likeplasmid (harbouring the BAN(1-35)+H156Y+A181T+N190F+I201F+A209V+Q264Smutations in the gene encoding the amylase from SEQ ID NO: 4).

The 440 bp fragment is purified from an agarose gel and used as aMega-primer together with primer 113711 in a second PCR carried out onthe same template.

The resulting approximately 630 bp fragment is digested with restrictionenzymes EcoR V and Acc65 I and the resulting approximately 370 bp DNAfragment is purified and ligated with the pDN1528-like plasmid digestedwith the same enzymes. Competent Bacillus subtilis SHA273 (amylase andprotease low) cells are transformed with the ligation and Chlorampenicolresistant transformants are checked by DNA sequencing to verify thepresence of the correct mutations on the plasmid.

Primer 27274: (SEQ ID NO:31) 5′CATAGTTGCCGAATTCATTGGAAACTTCCC 3′ Primer1B: (SEQ ID NO:32) 5′CCGATTGCTGACGCTGTTATTTGC 3′ primer AM115: (SEQ IDNO:33) 5′GCCAAGCGGATAACGGCTACGGTGC 3′

Construction of LE314: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+A52S is carried our in a similarway, except that mutagenic primer AM116 is used.

AM116:

-   5′ GAACGAGCCAATCGGACGTGGGCTACGG 3′ (SEQ ID NO: 34)

Construction of LE315: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+A52S+V54N is carried our in asimilar way, except that mutagenic primer AM117 is used.

AM117:

-   5′ GGAACGAGCCAATCGGATAACGGCTACGGTGC 3′ (SEQ ID NO: 35)

Construction of LE316: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+T49L is carried our in a similarway, except that mutagenic primer AM118 is used.

AM118:

-   5′ GCATATAAGGGACTGAGCCAAGCGG 3′ (SEQ ID NO: 36)

Construction LE317: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+T49L+G107A is carried our in asimilar way, except that mutagenic primer AM118 and mutagenic primerAM119 are used simultaneously.

AM119:

-   5′ CAACCACAAAGCCGGCGCTGATGCG 3′ (SEQ ID NO: 37)

Construction of LE318: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+A52S+V54N+T49L+G107A is carried ourin a similar way, except that mutagenic primer AM120 and mutagenicprimer AM119 are used simultaneously.

AM120:

-   5′ GCATATAAGGGACTGAGCCAATCGGATAACGGCTACGGTGC 3′ (SEQ ID NO: 38)

Construction of LE 319: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+A52S+V54N+T49L is carried our in asimilar way, except that mutagenic primer AM120 is used.

Construction of LE320: BAN/Termamyl hybrid+H156Y+A181T+N190F+A209V+Q264S+G107A is carried our in a similar way, except that mutagenic primerAM119 is used.

Construction of LE322: BAN/Termamyl hybrid+H156Y+A181T+N190F+A209V+Q264S+Q51 R+A52S is carried our in a similar way, except that mutagenicprimer AM121 is used.

AM121:

-   5′ GAACGAGCCGATCGGACGTGGGCTACGG 3′ (SEQ ID NO:39)

Construction of LE323: BAN/Termamylhybrid+H156Y+A181T+N190F+A209V+Q264S+A52N is carried our in a similarway, except that mutagenic primer AM122 is used.

AM122:

-   5′ GAACGAGCCAAAACGACGTGGGCTACGG 3′ (SEQ ID NO: 40)

Example 3

Testing of LE429 Variants (Saccharification)

The standard reaction conditions were:

Substrate concentration 30% w/w Temperature 60° C. Initial pH (at 60°C.) 5.5 Enzyme dosage Glucoamylase 0.18 AGU/g DS Pullulanase 0.06 PUN/gDS Alpha-amylase 10 micro g enzyme/g DSDextrozyme™ E was used to provide glucoamylase and pullulanaseactivities

Substrates for saccharification were prepared by dissolving common cornstarch in deionized water and adjusting the dry substance toapproximately 30% w/w. The pH was adjusted to 5.5 (measured at 60° C.),and aliquots of substrate corresponding to 10 g dry weight weretransferred to blue cap glass flasks.

The flasks were then placed in a shaking water bath equilibrated at 60°C., and the enzymes added. The pH was readjusted to 5.5 where necessary.The samples were taken after 48 hours of saccharifcation; the pH wasadjusted to about 3.0, and then heated in a boiling water bath for 15minutes to inactivate the enzymes. After cooling, the samples weretreated with approximately 0.1 g mixed bed ion exchange resin (BIO-RAD501 X8 (D)) for 30 minutes on a rotary mixer to remove salts and solubleN. After filtration, the carbohydrate composition was determined byHPLC. The following results were obtained:

The parent alpha-amylase for the variants is LE429.

Added SPEC. Alpha-amylase ACT. Variants DP₁ DP₂ DP₃ (NU/mg) V54N 96.11.75 1.18 8200 A52S 95.9 1.80 1.11 18800 A52S + V54N 96.3 1.84 1.0810000 T49L 96.3 1.77 1.11 12300 T49L + G107A 96.4 1.87 0.72 13600 A52S +V54N + T49L + G107A 80.5 2.55 0.43 10000 A52S + V54N + T49L 95.8 1.760.84 8400 G107A 94.4 1.89 1.04 19600 Q51R + A52S 95.9 1.77 1.27 16500A52N 95.5 1.89 1.56 17600 LE174 (CONTROL) 95.9/ 1.87/ 1.17/ 16000 95.81.83 1.35

Compared with the control, the presence of an active alpha-amylasevariant of the invention during liquefaction results in decreased panoselevels (DP3).

Especially the T49L+G107A variant of LE429 and the A52S+V54N+T49Lvariant of LE429, respectively, result in a drastically decreased panoselevel (DP₃). If these alpha-amylase variants are used for starchliquefaction, it will not be necessary to inactivate the enzyme beforethe commencement of saccharification.

Example 4

Liquefaction and Saccharification of LF429 Variants

The experiment in Example 3 was repeated for a number of other LE429variants under the same conditions.

The result is shown below:

Variant/sugar profile DP1 DP2 DP3 DP4+ T49V + G107A 95.9% 1.72% 1.27%1.11% T49Y + G107A 95.3% 1.73% 1.29% 1.65% T49N + G107A 95.7% 1.64%1.51% 1.18% T49L + A52S + G107A 95.7% 1.73% 0.95% 1.67% T49L + A52T +G107A 95.8% 1.66% 1.03% 1.48% T49L + A52F + G107A 95.7% 1.69% 1.16%1.42% T49L + A52L + G107A 95.5% 1.70% 1.40% 1.38% T49L + A52I + G107A95.9% 1.72% 1.31% 1.07% T49L + A52V + G107A 94.7% 1.69% 1.16% 2.44%T49L + A52V + G107A + A111V 94.5% 1.75% 0.72% 2.99% LE429 94.9% 1.71%1.85% 1.51%

Example 5

The experiment in Example 3 was repeated for a number of LE429 variants,except that the liquefaction was carried out at 95? C, pH 6.0 and thesaccharification at 60? C, pH 4.5, 40 ppm CaCl₂, followed byinactivation. The variant referred to below are LE429 variant. Theresults found are as follows:

Variant/sugar profile DP4+ DP3 DP2 DP1 T49F 1.15 0.92 1.83 96.12 T49D +G107A 0.84 1.03 1.82 96.3 T49I + G107A 0.97 0.64 1.84 96.55 T49L + G107A0.96 0.81 1.82 96.42 T49L + A52S + G107A 1.37 0.75 1.88 96.01 T49L +A52T + G107A 0.87 0.81 1.8 96.52 T49L + A52F + G107A 0.98 0.83 1.8796.31 T49V + G107A 0.65 0.8 2.13 96.43 T49Y + G107A 0.83 0.94 1.89 96.35LE429 1.16 1.21 1.77 95.87References Cited

-   Klein, C., et al., Biochemistry 1992, 31, 8740-8746,-   Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283,-   Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238,-   Larson, S. B., J. Mol. Biol. (1994) 235,1560-1584,-   Lawson, C. L., J. Mol. Biol. (1994) 236, 590-600,-   Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799,-   Brady, R. L., et al., Acta Crystallogr. sect. B, 47, 527-535,-   Swift, H. J., et al., Acta Crystallogr. sect. B, 47, 535-544-   A. Kadziola, Ph.D. Thesis: “An alpha-amylase from Barley and its    Complex with a Substrate Analogue Inhibitor Studied by X-ray    Crystallography”, Department of Chemistry University of Copenhagen    1993-   MacGregor, E. A., Food Hydrocolloids, 1987, Vol.1, No. 5-6, p.-   B. Diderichsen and L. Christiansen, Cloning of a maltogenic amylase    from Bacillus stearothenmophilus, FEMS Microbiol. letters: 56: pp.    53-60 (1988)-   Hudson et al., Practical Immunology, Third edition (1989), Blackwell    Scientific Publications,-   Sambrook et al., Molecular Cloning-A Lahoratory Manual, 2nd Ed.,    Cold Spring Harbor, 1989-   S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981,    pp. 1859-1869-   Matthes et al., The EMBRO. J. 3, 1984, pp. 801-805.-   R. K. Saiki et al., Science 239, 1988, pp. 487-491.-   Morinaga et al., (1984, Biotechnology 2:646-639)-   Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151-   Hunkapiller et al., 1984, Nature 310:105-111-   R. Higuchi, B. Krummel, and R. K. Saiki (1988). A general method of    in vitro preparation and specific mutagenesis of DNA fragments:    study of protein and DNA interactions. Nuci. Acids Res.    16:7351-7367.-   Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.-   Gryczan et al., 1978, .J. Bacteroil. 134, pp. 318-329.-   S. D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682.-   Boel et al., 1990, Biochemisry 29, pp. 6244-6249.-   Sarkar and Sommer, 1990, BioTechniques 8, pp. 404-407.

1. A variant of a parent alpha-amylase, wherein said variant has anamino acid sequence which has at least 90% homology to the amino acidsequence shown in SEQ ID NO:2, the amino acid sequence shown in SEQ IDNO:4, the amino acid sequence shown in SEQ ID NO:6, or the amino acidsequence shown in SEQ ID NO:8, wherein said variant has alpha-amylaseactivity and wherein said variant comprises an alteration at a positioncorresponding to one or more of positions G48, T49 and G107 (using SEQID NO:4 for numbering).
 2. The variant of claim 1, wherein said variantcomprises an alteration at a position corresponding to position G48 inSEQ ID NO:4.
 3. The variant of claim 1, wherein said variant comprisesan alteration at a position corresponding to position T49 in SEQ IDNO:4.
 4. The variant of claim 1, wherein said variant comprises analteration at a position corresponding to position G107 in SEQ ID NO:4.5. The variant of claim 1, wherein said variant comprises an alterationof G48A, V, S, T, or I.
 6. The variant of claim 1, wherein said variantcomprises an alteration of G48A.
 7. The variant of claim 1, wherein saidvariant comprises an alteration of T49V, I, D, N, Y, F, W, M, E Q K, orR.
 8. The variant of claim 1, wherein said variant comprises analteration of G107A, V, S, T, I, L, C.
 9. The variant of claim 1,wherein said variant comprises an alteration of G107A.
 10. The variantof claim 1, wherein said variant has an amino acid sequence which has atleast 90% homology to the amino acid sequence shown in SEQ ID NO:2. 11.The variant of claim 1, wherein said variant has an amino acid sequencewhich has at least 95% homology to the amino acid sequence shown in SEQID NO:2.
 12. The variant of claim 1, wherein said variant has an aminoacid sequence which has at least 97% homology to the amino acid sequenceshown in SEQ ID NO:2.
 13. The variant of claim 1, wherein said varianthas an amino acid sequence which has at least 99% homology to the aminoacid sequence shown in SEQ ID NO:2.
 14. The variant of claim 1, whereinsaid variant has an amino acid sequence which has at least 90% homologyto the amino acid sequence shown in SEQ ID NO:4.
 15. The variant ofclaim 1, wherein said variant has an amino acid sequence which has atleast 95% homology to the amino acid sequence shown in SEQ ID NO:4. 16.The variant of claim 1, wherein said variant has an amino acid sequencewhich has at least 99% homology to the amino acid sequence shown in SEQID NO:4.
 17. The variant of claim 1, wherein said variant has an aminoacid sequence which has at least 90% homology to the amino acid sequenceshown in SEQ ID NO:6.
 18. The variant of claim 1, wherein said varianthas an amino acid sequence which has at least 95% homology to the aminoacid sequence shown in SEQ ID NO:6.
 19. The variant of claim 1, whereinsaid variant has an amino acid sequence which has at least 99% homologyto the amino add sequence shown in SEQ ID NO:6.
 20. The variant of claim1, wherein said variant has an amino acid sequence which has at least90% homology to the amino acid sequence shown in SEQ ID NO:8.
 21. Thevariant of claim 1, wherein said variant has an amino acid sequencewhich has at least 95% homology to the amino acid sequence shown in SEQID NO:8.
 22. The variant of claim 1, wherein said variant has an aminoacid sequence which has at least 99% homology to the amino acid sequenceshown in SEQ ID NO:8.
 23. A variant of a parent aipha-amylase, saidvariant having alpha-amylase activity and comprising the amino acidsequence shown in SEQ ID NO:4, wherein the N-terminal 35 amino acidresidues of the amino acid sequence shown in SEQ ID NO:4 are replacedwith the N-terminal 33 amino acid residues of the amino acid sequenceshown in SEQ ID NQ:6.
 24. The variant of claim 23, wherein said variantfurther comprises the following mutations H156Y+A181T+N190F+A209V+Q264S.